PROJECT SUMMARY / ABSTRACT Diabetes profoundly impairs the tissue repair process, leading to chronic non-healing wounds, which represent a leading cause of lower limb amputations. The role of vascular pathology in impaired diabetic wound healing (“under healing”) has been well established, and the role of external mechanical forces across wounds in promoting excessive scar formation (“over healing”) is similarly well studied. However, the mechanisms through which these countervailing systems interact within diabetic tissue to yield non-healing skin ulcers have yet to be thoroughly examined. During prior years of NIH-funded research, important contributions have been made to our knowledge of the critical role of vascular progenitor cells in normal and diabetic wound healing. These include the first studies on single cell analysis of diabetic subpopulations during wound healing in both mice and humans, which identified specific cell subtype depletions that contribute to impaired blood vessel formation and delayed healing. More recently, the role of mechanoresponsive fibroblast populations in driving excessive skin scarring and ineffective wound closure has been examined in similar pathologic states. To understand the effects of diabetes and mechanical force on cell population dynamics with greater precision, we have developed novel single cell techniques to identify critical perturbations in cell subpopulations. In this proposal, we will apply these emerging -omics technologies to characterize the behavior of cell populations in non-healing diabetic wounds. It is our fundamental hypothesis that local tissue mechanical forces contribute to the disruption of cellular ecology in diabetic wound healing and that mitigation of these forces can improve healing. To achieve this, we will first employ a novel multiplex approach to high-throughput single cell sequencing to evaluate changes to cell populations in human diabetic wounds healing under different mechanical environments (Specific Aim 1). We will then confirm the changes in human diabetic cell populations using animal models, while more precisely assessing the effect of skin tension on healing kinetics (Specific Aim 2), which will further clarify the functional role of these cells. Finally, we will use real world data (RWD) from electronic health records to evaluate the efficacy of therapies aimed at offsetting mechanical forces, in order to develop clinical models to guide treatment strategies (Specific Aim 3). Collectively, this work will enhance our understanding of diabetic wound biology and its interaction with the external mechanical environment, paving the way for future therapeutic approaches, while also providing generalizable clinical recommendations for force offloading therapies that can be readily applied to guide treatment decisions at wound centers across the United States. The studies described in this proposal reflect the multi-faceted approach to translational medical research that...